A 25-year-old unvaccinated mare has died from hendra virus near Scone.
According to Local Land Services, the horse developed neurological signs on Friday, after being confined to a yard for three days.
It was later euthanised by the owners after becoming unresponsive.
A district veterinarian from Hunter Local Land Services visited the property on Sunday to collect samples with results on Wednesday from the State Veterinary laboratory at Menangle confirming hendra virus infection.
No other horses on the property showed signs of illness and NSW health is undertaking risk assessments on any impacted humans.
Most hendra cases in NSW have been on the north coast, with a case at Kempsey in 2013 the previous most southern occurrence.
The state has recorded 22 horse deaths to hendra since the first case in 2006.
The Australian Veterinary Association had been advised of the latest case by the NSW Department of Primary Industries.